The broad long-term objective of this proposal is to elucidate the mechanism of globin gene regulation by the erythroid transcription factor GATA-1. The specific aims are to identify amino acid residues encoding DNA binding specificity and activation of transcription, clarify the role of binding affinity in the function of specific GATA-1 sites, and characterize the effect of interactions of GATA-1 with other transcription factors on promoter activity and developmental specificity in erythroid cells. We will continue site-directed mutagenesis studies to identify the amino acid residues directing sequence specificity and stability of DNA binding. Fusion of GATA-1 domains to the DNA binding domain of GAL will be tested in erythroid cell lines for the ability to activate transcription and interact with other transcription factors. Mutants of GATA-1 will be used to reconstitute erythropoiesis derived from an ES cell line lacking a functional GATA-1 gene. GATA sites selected from the human beta-globin locus will be assayed for binding affinity, and sites with differing affinities will be compared for activity and developmental specificity. We will construct minimal erythroid promoters from GATA-1, CACCC, and AP- 1/NFE-2 binding sites, and assay their activity and developmental specificity in erythroid cell lines. Cis-elements binding other transcription factors will be linked to GATA-1 sites in minimal promoters and tested for interaction in transient assays. Erythroid "minigenes" will be constructed from oligonucleotides encoding GATA-1 sites and interacting cis-elements flanked by matrix attachment regions (MARs), and driving expression of LacZ. These will be tested in transgenic mice for developmental stage specificity.